Method for the stabilization of chimeric immunoglobulins or immunoglobulin fragments, and stabilized anti-EGP-2 scFv fragment

ABSTRACT

The present invention relates to a method for stabilizing chimeric immunoglobulins or immunoglobulin fragments. Furthermore, the invention also provides a stabilized anti-EGP-2 scFv fragment.

This application is a continuation of PCT/EP00/03176 filed Apr. 10,2000.

The present invention relates to a method for stabilizing chimericimmunoglobulins or immunoglobulin fragments. Furthermore, the inventionalso provides a stabilized anti-EGP-2 scFv fragment.

Small antibody fragments show exciting promise for use as therapeuticagents, diagnostic reagents, and for biochemical research. Thus, theyare needed in large amounts, and the expression of antibody fragments,e.g. Fv, single-chain Fv (scFv), or Fab in the periplasm of E. coli(Skerra & Plückthun 1988; Better et al., 1988) is now used routinely inmany laboratories. Expression yields vary widely, however, especially inthe case of scFvs. While some fragments yield up to several mg offunctional, soluble protein per liter and OD of culture broth in shakeflask culture (Carter et al., 1992, Plückthun et al. 1996), otherfragments may almost exclusively lead to insoluble material, often foundin so-called inclusion bodies. Functional protein may be obtained fromthe latter in modest yields by a laborious and time-consuming refoldingprocess. The factors influencing antibody expression levels are stillonly poorly understood. Folding efficiency and stability of the antibodyfragments, protease lability and toxicity of the expressed proteins tothe host cells often severely limit actual production levels, andseveral attempts have been tried to increase expression yields. Forexample, Knappik & Plückthun (1995) have identified key residues in theantibody framework which influence expression yields dramatically.Similarly, Ullrich et al. (1995) found that point mutations in the CDRscan increase the yields in periplasmic antibody fragment expression.Nevertheless, these strategies are only applicable to a few antibodies.

The observations by Knappik & Plückthun (1995) indicate that optimisingthose parts of the antibody fragment which are not directly involved inantigen recognition can significantly improve folding properties andproduction yields of recombinant Fv and scFv constructs. The causes forthe improved expression behaviour lie in the decreased aggregationbehaviour of these molecules. For other molecules, fragment stabilityand protease resistance may also be affected. The understanding of howspecific sequence modifications change these properties is still verylimited and currently under active investigation.

Single-chain Fv fragments (scFvs) are recombinant antibody fragmentsconsisting of the variable domains of the heavy and light chain,connected by a flexible peptide linker¹² ¹³. These fragments conservethe monovalent binding affinity and the specificity of the parent mAband can be efficiently produced in bacteria¹⁴. ScFvs can be constructedby cloning the variable domains of a mAb showing interesting bindingproperties from hybridoma cells or by direct selection of scFv fragmentswith the desired specificity from immunized or naive phagelibraries^(15,16). Frequently scFvs cloned from hybridomas show poorproduction yields and low thermodynamic stability which limit theirusefulness for in vivo applications¹⁷, whereas scFvs selected from phagelibraries have already undergone selection not only for antigen binding,but also for stability and folding properties in the scFv format¹⁸.

For therapeutic applications, human antibodies or antibody fragments arepreferred to avoid an immune response e.g. against a murine antibodyfragment derived from a monoclonal antibody (HAMA response). To solvethat problem, human antibody fragments can be obtained by screeninghuman antibody libraries (EP-A1 0 859 841; Vaughan et al., 1996).Another solution is to transplant the specificity of a non-humanmonoclonal antibody by grafting the CDR regions onto a human framework(EP-B1 0 239 400). In an improvement of said technique, humanizedantibodies or antibody fragments with improved binding behavior can beproduced by incorporating additional residues derived from saidnon-human antibody (EP-B1 0 451 216). In addition to achievinghumanization, these techniques allow to “repair” scFv fragments withsuboptimal stability and/or folding yield by grafting of the CDRs of ascFv fragment with the desired binding affinity and specificity onto theframework of a different, better behaved scFv, as was shown for thefluorescein binding antibody fragment 4-4-20 whose CDRs were grafted onthe 4D5-framework, leading to a clear improvement of both expressionyield and thermodynamic stability¹⁸. The 4D5 framework itself is anartificial framework resulting from the human consensus sequence and wasused for the humanization of the anti-c-erbB2 (p185^(Her 2)-ECD) 4D5 mAb(Herceptin™)¹⁹. Later studies showed the above average thermodynamicstability of the 4D5 antibody fragment²⁰, which correlates to thethermal stability of this molecule (Wörn and Plückthun, 1999) and isapparently of general importance for the in vivo application of scFvs.

The murine monoclonal antibody (mAb) MOC31 recognizes the 38 kDatransmembrane epithelial glycoprotein-2¹ (EGP-2; also known as GA733-2,Ep-CAM or KSA). EGP-2 is regarded as a suitable target antigen for tumorimaging and therapy, since it is highly overexpressed on a variety ofhuman carcinomas and is not shed into the circulation. Several clinicaltrials with anti-EGP-2 mAbs such as 17-1A, KS1/4 and MOC31^(2,3,4)demonstrated the potential of these antibodies for active and passiveimmunotherapy of human carcinomas. The exact function of thetransmembrane glycoprotein EGP-2 is not yet known, although a role incell-cell association has been proposed (Simon et al., 1990). Recentreports identify EGP-2 as a homophilic cell-cell adhesionmolecule^(5,6), and EGP-2 has been identified as a potential modifier ofinvasiveness and chemoresponsiveness⁷. In a study evaluating thepotential of new immunotherapeutics targeted to EGP-2, exotoxin-A (ETA)chemically fused to mAb MOC31 was found to retard the growth of largetumors⁸.

Carcinoma-associated antigens such as c-erbB2 and EGF-receptor, as wellas EGP-2 have served as targets for radiolabelled antibodies for tumorimaging and therapy. Effort have been made to improve the targetingefficiency by reducing the molecular weight and thereby increasing thetissue penetration and serum clearance of such antibody-basedconstructs. Fab, (Fab)², dsFv and scFv fragments, generated byrecombinant antibody technology, have great potential in thisrespect^(9,10), although up to now the optimal formats concerningstability, molecular weight and affinity have not been determined andhave to be fine-tuned for the different antibody-effector fusionproteins depending on the special in vivo system and application goal¹¹.

For the development of new antibody fragment based imaging andtherapeutic reagents directed to the pancarcinoma associated antigenepithelial glycoprotein-2 the variable domains of the murine anti-EGP-2hybridoma MOC31 was cloned in the single-chain Fv fragment format¹⁶.Although the resulting scFv showed the expected binding affinity andspecificity towards EGP-2, which was also shown on tissues sections inimmunohistostaining experiments by others³⁰, it was poorly expressed inthe periplasm of bacteria. In vivo targeting experiments in nude miceemploying this scFv fragment failed. The scFv not only did notaccumulate in the tumor, but also showed slower clearance rates than anirrelevant control scFv directed against fluorescein. It could be shownthat the MOC31 scFv formed high molecular weight aggregates and rapidlylost its activity when incubated in serum at body temperature (37° C.).This was primarily due to insufficient thermal stability rather thanproteolytic degradation, since similar precipitation and loss ofimmunoactivity could also be observed upon incubation of highly purifiedscFv in PBS at 37° C.

To derive from this aggregation-prone and thermally instable scFv amolecule suitable for immunotherapeutic application, the biophysicalproperties of the construct had to be improved. Basically, two avenueswere open to approach this goal: In-vitro evolution of the MOC31 scFvtowards better thermal stability by combining randomization withselection for improved functionality³⁵ at elevated temperature or thetransfer of the binding specificity of the anti-EGP-2 scFv MOC31 onto ascFv framework with above average biophysical properties by CDRgrafting¹⁸. Although the first option has been successfully used toachieve extremely stable scFvs³⁵, the second option had the addedadvantage that by choosing a human framework sequence for the graft, ahumanization could be achieved at the same time, thus reducing thepotential immunogenicity of future immunotherapeutic reagents. It wastherefore decided to graft the anti-EGP-2 scFv MOC31 binding specificityonto the artificial human consensus framework of scFv 4D5, essentiallycorresponding to the germline sequences IGVH 3-66 and IGVK 1-39 (IMGT).Grafting of complementary determining regions (CDRs) of mAbs forhumanization has been used more than 100 times for humanization¹⁰ andcan now be regarded as a standard technology. The 4D5 framework has beenused successfully several times before as an CDR acceptor^(21,18,36).

This strategy proved successful, since the graft variant 4D5MOC-A showedbinding characteristics indistinguishable to those of the parentantibody and scFv.

However, 4D5MOC-A showed only a thermal stability intermediate betweenthat of the two parent molecules 4D5 and MOC31.

Biodistribution data indicated, that scFv MOC31, which lost most of itsactivity within less than 1 hour at 37° C. failed to enrich at thetumor, the graft variant 4D5MOC-A, stable for a few hours at 37° C.enriched only slightly.

Thus, the technical problem underlying the present invention is toprovide a method which enables the stabilization of chimericimmunoglobulins or immunoglobulin fragments formed by CDR-graftingapproaches. A further technical problem underlying the present inventionis to stabilize the chimeric anti-EGP-2-binding scFv fragment 4D5MOC-A.The solution to the above technical problems is achieved by theembodiments characterized in the claims. Accordingly, the presentinvention allows to identify and modify residues of the chimericimmunoglobulins or immunoglobulin fragments which lead to increasedstability. The technical approach of the present invention, i.e. theidentification and exchange of amino acid residues in the frameworkregions of the VH domain to stabilize chimeric immunoglobulins orimmunoglobulin fragments formed by CDR-grafting approaches is neitherprovided nor suggested by the prior art.

Thus, the present invention relates to a method for stabilizing achimeric immunoglobulin or immunoglobulin fragment (chimera) being ableto bind to an antigen, wherein said chimera comprises a VH and a VLdomain comprising

-   -   i) antigen-binding loops derived from a donor immunoglobulin or        immunoglobulin fragment (donor) which is able to bind to said        antigen, and    -   ii) framework regions derived from an acceptor immunoglobulin or        immunoglobulin fragment (acceptor), and, optionally,    -   iii) further residues from said donor if required for improving        antigen-binding,        and wherein the VH domains of said donor and of said acceptor        belong to different framework structure subgroups, said method        comprising the steps of    -   a) comparing the structural features of the VH do mains of said        acceptor and of said donor;    -   b) identifying one or more framework positions in VH where        different amino acid residues present in said acceptor and said        donor lead to the formation of different framework structure        subgroups; and    -   c) setting up a stabilized antigen-binding immunoglobulin or        immunoglobulin fragment by replacing in the chimeric one or more        of the residues present at said positions in the acceptor by        those present at said positions in the donor.

In the context of the present application, the following abbreviationsare used:

“Chimera” is used instead of the expression “chimeric immunoglobulin orimmunoglobulin fragment”, “donor” instead of “donor immunoglobulin orimmunoglobulin fragment”, and “acceptor” instead of “acceptorimmunoglobulin or immunoglobulin fragment”. The term “chimeric” in thecontext of the present invention refers to a molecule composed ofportions from two different molecules.

Immunoglobulin fragments according to the present invention may be Fv,scFv, disulfide-linked Fv (Glockshuber et al., 1992; Brinkmann et al.,1993), Fab, (Fab′)₂ fragments or other fragments well-known to thepractitioner skilled in the art, which comprise the variable domain ofan immunoglobulin or immunoglobulin fragment.

Particularly preferred is the scFv fragment format.

The term “antigen-binding loops” refers to those parts of the variabledomain of immunoglobulins or immunoglobulin fragments which areprimarily responsible for antigen-binding. Kabat et al. (1979) definedcomplementarity determining regions (CDRs) as being responsible forantigen-binding based on the degree of variability found in antibodysequences. Later, Chothia and co-workers defined the antigen-bindingloops based on structural considerations. Allazikani et al. (1997)review and compare the definitions according to Kabat and Chothia. Theterm “further residues from said donor if required” refers to thesituation that additional residues outside of the antigen-binding loopsare grafted onto the acceptor. EP-B1 0 451 216 teaches methods whichallow to identify such further residues.

The analysis according to the present invention involves the analysis ofcontacts between framework residues and the identification ofdifferences in hydrogen-bonding patterns, torsion angles of side chains,changes of the polypeptide main chain conformation and of the tertiarystructure. Particularly preferred is the analysis of the residues inframework 1 of the VH domain, and most preferred the analysis ofdifferences caused by different residues in positions H6 to H10, and theconsequences of such differences throughout the VH domain byinteractions with further VH domain residues and correlated sequence andconformational differences³¹. Differences in positions H6 to H10 can beused to define different framework structure subgroups.

In the context of the present invention, a numbering scheme is used inaccordance with Kabat et al. (1979). Thus, the number does notnecessarily correspond to the actual position in the sequential order ofresidues in a VH or VL chain, but indicates a relative positioncorresponding to the sequences in the Kabat database of antibodysequences. “H” refers to positions in VH, “L” to positions in VL. Thus,H6 is residue number 6 according to Kabat in VH.

In a preferred embodiment, the method of the present invention furthercomprises that step a) is performed by analyzing VH domain structuresand/or structure models. Data on VH domain structures can be obtainedfrom NMR studies, or preferably from X-ray structures of immunoglobulinsor immunoglobulin fragments. Homology models can be generated by usingdifferent molecular modelling software packages available and well-knownto the practitioner skilled in the art. Preferably, the molecularmodelling software Insight97 (Biosym/MSI, modules Homology, Biopolymerand Discover) is used. Preferably, the sequence identity of VH domainsused for structure analysis and/or structure modelling show a highdegree of sequence identity to the corresponding VH domain of said donoror acceptor. Preferably, said sequence identity is larger than 75%, andmost preferably, larger than 80%.

Further preferred is a method, wherein said one or more frameworkpositions comprise H6.

Yet further preferred is a method, wherein said one or more frameworkpositions comprise H9.

In another embodiment, the present invention relates to a method,wherein said acceptor is the human anti-c-ErbB2 scFv fragment 4D5(SEQ-ID No. 1).

The anti-c-ErbB2 scFv fragment 4D5 has been described hereinabove.

In a further preferred embodiment, said donor is the anti-EGP-2 scFvfragment obtained from the murine hybridoma MOC31 (SEQ-ID No. 2).

The murine hybridoma MOC31 and the anti-EGP-2 scFv fragment obtainedtherefrom is described hereinabove and in the example.

In a yet further preferred embodiment of the present invention, said oneor more framework positions in VH are H6, H9, H18, H20, H38, H63, H82and H109 (numbering according to Kabat et al. (1979), see above).

In a still further embodiment, the present invention relates to amethod, wherein said stabilized antigen-binding immunoglobulin orimmunoglobulin fragment is the is the anti-EGP-2 scFv fragment 4D5MOC-B(SEQ-ID No. 3).

In another embodiment, the present invention relates to anantigen-binding immunoglobulin or immunoglobulin fragment stabilizedaccording to a method of the present invention.

In a most preferred embodiment, said antigen-binding immunoglobulin orimmunoglobulin fragment is the anti-EGP-2 scFv fragment 4D5MOC-B (SEQ-IDNo. 3).

In yet another embodiment, the present invention relates to a modifiedantigen-binding immunoglobulin or immunoglobulin comprising the variabledomain of an antigen-binding immunoglobulin or immunoglobulin fragmentstabilized according to a method of the present invention, saidmodification being

-   a) the conversion to a different immunoglobulin fragment or full    immunoglobulin, and/or-   b) the attachment of additional moieties, such as detection or    purification tags, reporter molecules, effector molecules,    association domains or combinations thereof.

Said modified immunoglobulin fragments according to the presentinvention may be an Fv, scFv, disulfide-linked Fv, Fab, (Fab′)₂fragments or other fragment, or a full immunoglobulin such as IgG, IgA,IgM well-known to the practitioner skilled in the art, which comprisethe variable domain of said stabilized immunoglobulin or immunoglobulinfragment and which is different from the immunoglobulin orimmunoglobulin fragment format of said stabilized immunoglobulin orimmunoglobulin fragment.

Particularly preferred are moieties which have a useful therapeuticfunction. For example, the additional moiety may be a toxin moleculewhich is able to kill cells (Vitetta et al., 1993). There are numerousexamples of such toxins, well known those skilled in the art, such asthe bacterial toxins Pseudomonas exotoxin A, and diphtheria toxin, aswell as the plant toxins ricin, abrin, modeccin, saporin, and gelonin.By fusing such a toxin to an immunoglobulin or immunoglobulin fragmentaccording to the present invention, the toxin can be targeted to, forexample, diseased cells, and thereby have a beneficial therapeuticeffect. Alternatively, the additional moiety may be a cytokine, such asIL-2 (Rosenberg & Lotze, 1986), which has a particular effect (in thiscase a T-cell proliferative effect) on a family of cells. In a furtherpreferred embodiment, the additional moiety is at least part of asurface protein which may direct the fusion protein to the surface of anorganism, for example, a cell or a phage, and thereby displays theimmunoglobulin or immunoglobulin fragment partner. Preferably, theadditional moiety is at least part of a coat protein of filamentousbacteriophages, most preferably of the geneIII protein. In a furtherembodiment, the additional moiety may confer on its immunoglobulin orimmunoglobulin fragment partner a means of detection and/orpurification. For example, the fusion protein could comprise themodified immunoglobulin or immunoglobulin fragment and an enzymecommonly used for detection purposes, such as alkaline phosphatase(Blake et al., 1984). There are numerous other moieties which can beused as detection or purification tags, which are well known to thepractitioner skilled in the art. Also provided for by the invention areadditional moieties such as the commonly used c-myc and FLAG tags (Hoppet al., 1988; Knappik & Plückthun, 1994).

By engineering one or more fused additional domains, immunoglobulin orimmunoglobulin fragment can be assembled into larger molecules whichalso fall under the scope of the present invention. To the extent thatthe physical properties of the immunoglobulin or immunoglobulin fragmentdetermine the characteristics of the assembly, the present inventionprovides a means of increasing the stability of such larger molecules.For example, mini-antibodies (Pack, 1994) are dimers comprising two scFvfragments, each fused to a self-associating dimerization domain.Dimerization domains which are particularly preferred include thosederived from a leucine zipper (Pack & Plückthun, 1992) orhelix-turn-helix motif (Pack et al., 1993).

All of the above embodiments of the present invention can be effectedusing standard techniques of molecular biology known to anyone skilledin the art.

In a further preferred embodiment, said modification is the attachmentof a penta- or hexa-histidin-tag.

Peptides comprising at least five histidine residues (Hochuli et al.,1988) are able to bind to metal ions, and can therefore be used for thepurification of the protein to which they are fused (Lindner et al.,1992). Vectors such as pIG-6 (see example) encoding a pentahistidin tailmay be used to produce such modified immunoglobulins or immunoglobulinfragments. In addition, pIG-6 provides an N-terminal FLAG-tag and aC-terminal c-myc-tag for detection purposes.

Further preferred is a modified fragment wherein said penta- orhexa-histidin-tag is complexed with a 99mTc-tricarbonyl moiety.

The His-tag specific 99mTc labeling method has been described (Albertoet al., 1998). 99mTc-tricarbonyl trihydrate forms very stable complexeswith the penta- or hexahistidine tag. The modified fragments containinga 99mTc-tricarbonyl moiety may be used for radiotherapy or radioimagingapproaches.

In a most preferred embodiment, the present invention relates to amodified fragment, comprising the variable domain of the anti-EGP-2 scFvfragment 4D5MOC-B (SEQ-ID No. 3) according to the present invention.

Further preferred are a nucleic acid sequence or nucleic acid sequencesencoding an antigen-binding immunoglobulin or immunoglobulin fragmentaccording to the present invention. Depending on the immunoglobulin orimmunoglobulin fragment type, a single nucleic acid sequence, e.g. forencoding an scFv fragment, or two, e.g. for encoding an Fab fragment, ormore nucleic acid sequences are required. Preferentially said nucleicacid sequences are comprised in a vector, preferably a vector suitablefor sequencing and/or expression. Said vector comprising said nucleicacid sequence or nucleic acid sequences may be comprised in a host cell.

In a further preferred embodiment, the present invention relates to amethod for the production of a stabilized antigen-binding immunoglobulinor immunoglobulin fragment according to the present invention comprisingthe expression of one or more nucleic acid sequences according to theinvention encoding said antigen-binding immunoglobulin or immunoglobulinfragment in a suitable expression system.

The expression system may be expression in a suitable host. The hostreferred to herein may be any of a number commonly used in theproduction of heterologous proteins, including but not limited tobacteria, such as E. coli (Ge et al, 1995), or Bacillus subtilis (Wu etal., 1993), fungi, such as yeasts (Horwitz et al., 1988; Ridder et al.,1995) or filamentous fungus (Nyyssönen et al., 1993), plant cells(Hiatt, 1990, Hiatt & Ma, 1993; Whitelam et al., 1994), insect cells(Potter et al., 1993; Ward et al., 1995), or mammalian cells (Trill etal., 1995). The expression system may be expression in a cell-freetranslation system, preferably in a coupled in vitrotranscription/translation system. Preferably, such a translation iscarried out in a prokaryotic translation system. Particularly preferredis an E. coli based translation system such as the S-30 E. colitranslation system. Alternatively, the translation may be carried out ina eukaryotic translation system.

In a further most preferred embodiment, the present invention relates toa pharmaceutical composition containing an antigen-bindingimmunoglobulin or immunoglobulin fragment according to the presentinvention and, optionally, a pharmaceutically acceptable carrier and/ordiluent.

In a yet further preferred embodiment, the present invention relates tothe use of a stabilized immunoglobulin or immunoglobulin fragmentaccording to the present invention, or of a modified immunoglobulin orimmunoglobulin fragment according to the present invention for thepreparation of a pharmaceutical composition for the treatment of humancarcinomas.

Further preferred is the use of the anti-EGP-2 scFv fragment 4D5MOC-B,or of a modified EGP-2-binding immunoglobulin or immunoglobulin fragmentaccording to the present invention for the preparation of apharmaceutical composition for the treatment of human carcinomas.

In a yet further preferred embodiment, the invention relates to adiagnostic composition containing an antigen-binding immunoglobulin orimmunoglobulin fragment according to the present invention.

In a still further preferred embodiment, the invention relates to adiagnostic kit containing an antigen-binding immunoglobulin orimmunoglobulin fragment according to the present invention.

FIGURE CAPTIONS

FIG. 1

Sequence alignment of the VL and VH domains of scFv MOC31, 4D5MOC-A,4D5MOC-B and 4D5: (A) Positions of sequence agreement between MOC31 and4D5 are indicated by black letters on a gray background, residues whichagree with 4D5 but are different from MOC31 by black letters on a whitebackground and residues which agree with MOC31 but not with 4D5 areindicated by white letters on a black background. Residue labels and CDRdefinitions are according to Kabat (1987). (B) indicates residues buriedin the domain core or interface, (b) semiburied residues. (•) and (•)indicate potential antigen contact residues, detected by a large(•, >40%) or small (•>1%) loss of side chain-solvent accessible surfaceupon complex formation, averaged for this position over all differentprotein-antibody complexes in the PDB database. Model of the scFvfragment 4D5MOC-B: (B) Quaternary structure of the anti-EGP-2 scFvfragment 4D5MOC-B, composed of VL (grey) and VH (white) with transferredpotential antigen contact residues of MOC31 (black). The eightadditional transferred murine residues in the core of VH are highlighted(black side chains). A space filling model shows 4D5MOC-B in the topview (C) and bottom view (D). Black balls are of murine and white of 4D5origin, while grey balls are the same in 4D5 and MOC31.

FIG. 2

Result of purification of scFv 4D5MOC-A and 4D5MOC-B. SDS-PAGE underreducing conditions shows the result of the purification of scFv4D5MOC-A and 4D5MOC-B after IMAC and subsequently performed ionexchange.

[MW standard :phosphorylase b (97.4 kDa); bovine serum albumine (66kDa); ovalbumine (44 kDa); carbonic anhydrase (29 kDa); trypsininhibitor (21.5 kDa); lysozyme (14.3 kDa)]

FIG. 3

Binding experiments with 99mTc-labelled 4D5MOC-A and -B (RIA).

(A) Competition RIA of Tc-labelled 4D5MOC-A and 4D5MOC-B on SW2-lungcancer cells with MOC31. Fifty ng radiolabelled scFv fragment wereincubated with or without MOC31 (10 μg) or with the same amount of ananti c-erbB2 monoclonal antibody as an irrelevant inhibitor.

(B) Binding specificity of 99mTc labelled 4D5MOC-A and 4D5MOC-B ondifferent antigens (500 ng/well).

FIG. 4

Check of thermal and serum Stability

Gel filtration over superdex 75 column before and after overnightincubation (20 h) at 37° C. of 4D5MOC-A (A) and 4D5MOC-B (B).Determination of remaining immunoactivity of ^(99m)Tc-labelledanti-EGP-2 scFv fragments before (C) and after overnight (D) incubationin human serum at 37° C. by incubation by Lindmo-assay²⁹.

The example illustrates the invention.

EXAMPLE (Willuda et al., 1999)

Material and Methods

Mammalian cell lines

The human small cell lung carcinoma cell line SW2, kindly provided byDr. S. D. Bernal (Dana Farber Cancer Institute, Boston, Mass., USA) andbreast cancer cell line SK-BR-3 (#HTB 30, American type culturecollection, Rockville, Md.) were maintained in RPMI 1640 (Hyclone,Europe Ltd.) based medium supplemented with 10% fetal calf serum (Gibco,Grand Island, N.Y.) and grown at 37° C. under an atmosphere of 5% CO₂.The breast cancer cell line SK-OV-3 (#HTB 77, American type culturecollection, Rockville, Md.) was grown in RPMI 1640, supplemented withEGF (10 ng/ml) and insulin (5 ng/ml).

Epithelial glycoprotein-2 (EGP-2) and Single-Chain Fv Fragments

The human epithelial glycoprotein-2 was produced as a recombinantsoluble protein (M1-F259) with an six-histidine C-terminal tag by use ofthe expression vector pBB4/GA-733-2 in the baculovirus expression system(Invitrogen). The anti-EGP-2 scFv fragment (scFv MOC31) was assembledfrom mRNA isolated from the murine hybridoma cell line MOC31¹ by using areengineered phage display system described before¹⁶. The single-chainFv fragment from the human anti-c-erbB2 antibody 4D5 had beenconstructed from the Fab fragment (Carter et al,) and had been used inseveral studies before^(21,20).

Molecular Modeling/Construction of Graft

A homology model of the anti-EGP-2 scFv fragment was generated using themolecular modelling software Insight97 (Biosym/MSI, modules Homology,Biopolymer and Discover). The V_(L) domain model was based on the x-raystructures of the mouse Fab fragment JEL103 (²², Brookhaven Databaseentries 1mrc, 1mrd, 1mre and 1mrf, 2.3‰–2.4‰ resolution, 76% sequenceidentity to MOC31), the V_(H) domain model was based on the structure ofan anti-neuraminidase Fab (^(23,24), pdb entries 1nca, 1ncb, 1ncc and1ncd, 2.5‰ resolution, 85% identity) and the CDR H3 conformation of theanti-choleratoxin Fab TE33 (²⁵, pdb entry 1tet, 2.3‰ resolution, 82%identity). The MOC31 domain models were superimposed on the crystalstructure of the Fv of humanized 4D5 version 8 (²⁶, pdb entry 1fvc, 2.2‰resolution, V_(L): 55% identity, V_(H): 50% identity to MOC31).Potential antigen contacts were identified by comparing the side chainsolvent accessible surface of known antibody-protein complexes in thepresence and absence of the ligand using the program naccess (S. Hubbardand J. Thornton, 1992, http://sjh.bi.umist.ac.uk/naccess.html). Themodels were checked for possible steric conflicts, potential antigencontacts and residues which might have an indirect influence on CDRconformations, resulting in the hybrid scFv 4D5MOC-A. In a secondconstruct, scFv 4D5MOC-B, 8 key residues in the core of V_(H) wereretained from the MOC31 sequence instead of changing them to the 4D5sequence in order to preserve the structural subtype of the MOC31 V_(H)framework.

The designed sequences for both variants were backtranslated(GCG-package) and the fragments were constructed by gene synthesis²⁷from eight overlapping oligonucleotides for V_(L) and ten for the twodifferent variants of V_(H), in the orientation V_(L)-linker-V_(H). Thelength of the used oligonucleotides was between 40 bp and 78 bp. Eachdomain was produced separately and cloned blunt-ended into thepBluescript vector (Stratagene) and was subsequently sequenced. V_(L)and V_(H) domains were then cloned into the expression vector pIG6 (Geet al., 1995). A 24-mer non-repetitive linker TPSHNSHQVPSAGGPTANSGTSGS²⁸was then introduced by cassette mutagenesis via AflII and BamHIrestriction sites.

Expression and Purification of Single-Chain Fv Fragments

For periplasmic expression of the c-erbB2 binding scFv fragment 4D5 andthe EGP-2 binding scFv fragments 4D5MOC-A and 4D5MOC-B, the pIG6 vectorwas used, while the pAK400 vector¹⁶ was used for the expression of scFvMOC31. For large scale production, the E. coli strain SB536 (Bass et al.1996) was used. One liter of dYT containing 1% glu and Ampicillin (30μg/ml) in a 5 l shake flask was inoculated with 30 ml of overnightculture. When the culture reached an OD_(550 nm) of 0.5, scFv productionwas induced with a final concentration of 1 mMisopropyl-D-galactopyranoside (IPTG; Boehringer Mannheim) for three tofour hours at 24° C. The final OD was five–six for 4D5, 4D5MOC-A4D5MOC-B and four for scFv MOC31. The harvested pellet was stored at−80° C.

For purification the pellet from 1 liter culture was re-suspended in 20mM Hepes pH 7.0, 30 mM NaCl and lysed with two cycles in a FrenchPressure Cell press (SLS instruments Inc., Urbana Ill., USA). Thecleared lysate was centrifuged in a SS-34 rotor at 48246 g at 4° C. andfilter sterilized. All single-chain Fv fragments were purified over aNi⁺-IDA-column and HS/M-4.6/100-ion exchange column, coupled in-line ona BIOCAD-System (Perseptive BioSystem-Inc.) as described previously(Plückthun 1996, book chapter). After loading the lysate on theNi⁺-IDA-column the column was washed with 20 mM Hepes pH 7.0, 500 mMNaCl, and in a second step with 20 mM Hepes pH 7.0, 40 mM imidazolebefore bound protein was eluted with 200 mM imidazole, pH 7.0. Theeluate was loaded directly on the HS/M-4.6/100-ion-exchange-column, andthe specifically bound protein was eluted with a salt gradient from 0 to500 mM NaCl in 20 mM Hepes pH 7.0. The fraction containing the antibodyfragment was dialed against an excess of PBS and concentrated to 1 mg/miusing a 10 kDa cut-off filter (Ultrafree-MC low protein binding,Millipore) by centrifugation at 4000 g at 4° C. For the anti-EGP-2 scFvfragment MOC31 it was necessary to perform a preparative gel filtrationover a Superdex75 column (Pharmacia) as the third purification stepwhich lowered the final yield again ten-fold. The result of thepurification was checked on a 12.5% SDS-PAGE under reducing conditions.The molecular weights of all purified scFvs were checked by massspectrometry.

His-tag Specific^(99m)Technetium Labeling of Single-Chain Fv Fragments

^(99m)Tc-tricarbonyl trihydrate (Alberto et al., 1998). forms verystable complexes with the penta- or hexahistidine tag, therby makingdual use of the His tag which is present for immobilized metal affinitychromatography (IMAC) purification. The scFv fragments (1 mg/ml) weremixed with one-third volume of ^(99m) technetium-tricarbonyl (30 mCi/ml)in buffer and one-third volume of 0.5 M MES pH 6.2 and incubated forthirty minutes at 37° C. ScFv MOC31 was labelled for 30 min at 30° C. ata protein concentration of 400 μg/ml to avoid precipitation. Thereaction mixture was desalted over a Fast desalting column (Pharmacia)equilibrated with PBS. Aliquots of the collected fractions were measuredin a scintillation counter to identify the fractions with the labelledprotein.

Analytical Gel Filtration

Analytical gel filtration was performed with the Smart system(Pharmacia), using a Superdex 75 column. All measurements were carriedout in PBS buffer containing 0.005% Tween-20. The scFv fragments wereinjected at a concentration of 1 mg/ml in a volume of 15 μl before andafter overnight incubation for 20 h at 37° C. The column was calibratedin the same buffer with alcohol dehydrogenase (150 kDa), bovine serumalbumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrom c (12.4 kDa)as molecular mass standards.

Binding Specificity

The binding specificity of the different scFv fragments was tested bycompetition with the monoclonal antibody MOC31. Fifty ng radiolabelledscFv 4D5MOC-A or 4D5MOC-B were incubated with 0.5×10⁶ SW2 cells in 200μl PBS/1% BSA after pre-incubation with or without the mAb MOC31 (10 μg)or with the same amount of an anti-c-erbB2 monoclonal antibody as anirrelevant competitor for 30 min at 40° C. In three washing steps cellswere centrifuged at 1000 g for 5 min at 40° C., the supernatantdiscarded and the cells re-suspended in PBS/1% BSA. The remainingradioactivity was then measured in a scintillation counter. In a furtherbinding experiment both scFv fragments (50 ng) were incubated withdifferent antigens, coated (500 ng/well) on a 96 well microtiter plate,to check for cross-reactivity. The wells were washed three times withPBS/1% BSA and the radioactivity was determined.

K_(D)-Determination by RIA and Surface Plasmon Resonance (BIAcore)

The binding affinity of the ^(99m)Tc labelled single-chain Fv fragmentswas determined on SW2 cells in a radio-immunoaffinity assay (RIA). SW2(0.5×10⁶) cells were incubated with increasing amounts of single-chainFv fragment (100 pM–30 nM) for 1 hour at 4° C. For estimation ofnonspecific binding control samples of cells were pre-incubated with a100-fold excess of unlabelled single-chain Fv fragment for 1 h at 40° C.The bound fraction of single-chain Fv fragment was determined in ascintillation counter. Each obtained value represents the mean of twosamples. Counts per minutes (cpm) were plotted against the nanomolarconcentration of single-chain Fv fragment and fitted with the non-linearregression function.

Kinetic rate constants were determined by surface plasmon resonance(SPR) with a BIAcore instrument. Recombinant soluble EGP-2-antigen wascovalently coupled to a CM-5 sensor chip via free amine groupsresulting, in a surface coverage of 350 resonance units. Single-chain Fvfragments were injected in increasing concentrations (0.1nM–4 μM) at aflow rate of 30 μl/min of degassed PBS/0.005% Tween-20. Association anddissociation rate constants were calculated from the sensorgram the by aglobal curve fit using the BIAevaluation 3.0 software (Pharmacia).

Serum Stability of Radiolabelled scFv at 37° C.

The fraction of single-chain Fv fragments remaining immunoactive afterradioactive labeling was determined as described previously²⁹. Samplescontaining different numbers of cells (0.625×10⁶–10×10⁶) were incubatedin 100 μl with fifty ng of radiolabelled scFv fragments for 1 h at 4° C.on a shaker. Unspecific binding was determined on control samples ofcells pre-incubated with a 100-fold excess of unlabelled scFv fragmentsin PBS/1% BSA. After three washing steps, the amount of bound scFvfragments was then determined in a scintillation counter. Each reportedvalue represents the mean of the result of two samples. For calculationof the immunoactivity total counts per minute (cpm) were divided bymeasured cpm value for bound protein and then plotted against inversecell number and fitted by linear regression. The inverse y-intercept inpercent gives the percentage bioactive single-chain Fv fragments. Toestimate the stability of the different radiolabelled single-chain Fvfragments in serum, the molecules were incubated overnight (20 h) inhuman serum at 37° C., at a final concentration of 17 μg/ml and theremaining immunoactivity determined in the Lindmo assay.

In Vivo Characterization—Clearance and Biodistribution

Blood clearance studies were performed with eight-week-old, tumor freefemale CD1 nude mice. Each mice received i.v. 300 μCi of ^(99m)Tclabelled scFv 4D5MOC-B. After 7.5, 15, 30, 60, 120, and 240 minutesfollowing injection, blood samples were taken, and t_(1/2)α and t_(1/2)βvalue was calculated from the measured radioactivity. Biodistributionstudy of ^(99m)Tc labelled scFv fragment 4D5MOC-B was done insix-week-old CD1 nude mice bearing 13 days old SW2 xenografts (40–80mg). Each mouse of three groups of four mice received 30 μgradiolabelled scFv (300 μCi). Biodistribution analysis of ^(99m)Tclabelled scFv MOC31 was performed in seven-week-old Black-nude mice(strain bl6 Uwe: Bl6??) carrying ten-day-old SW2-xenografts (10–40 mg).Each mouse of three groups of three mice was administered 5μg^(99m)technetium-labelled scFv MOC31 (85 μCi). Anti-fluoresceinbinding scFv FITC-E2 was used as an nonspecific control antibody. Themice were killed at 1, 4 and 24 hours after injection. Tissue and organswere removed and assessed for activity using a gamma-counter. Thebiodistribution analysis with ^(99m)Tc labelled scFv 4D5 was describedrecently (Waibel et al., 1999).

Results

Molecular Modeling—Construction of Graft

We have constructed the scFv fragment of MOC31 using standard phagedisplay methodology¹⁶, determined its functionality and demonstrated arather high affinity to its antigen EGP-2 (Table 1), consistent insequence and properties with an independently constructed scFv MOC31³⁰.Surprisingly, the in vivo localization of this scFv was hardlydistinguishable from a control scFv without EGP-2 specificity andessentially the scFv MOC31 did not localize to a xenografted tumor(table 2). We therefore hypothesized that this protein is not stableenough and designed two more stable variants, first, by grafting theloops to a well-characterized stable framework and second, additionallychanging several residues in the interior of one of the variabledomains. As the recipient framework, we chose the humanized version of4D5¹⁹, itself an product of a grafting exercise. This framework consistsessentially of a heavy chain variable domain derived from the germlineIGHV 3-66 (IMGT), VH 3-18 (Vbase), Locus DP 3-66 (DP-86) and a the kappalight chain variable domain derived from germline IGKV 1-39 (IMGT), VK1-1 (Vbase), locus DP O12.

A homology model of MOC31 was built and compared to the X-ray structureof the human 4D5 version 8 Fv fragment (pdb entry 1fvc). Potentialantigen contact residues were identified by an analysis ofantibody-protein antigen complexes in the Brookhaven Protein Database(FIG. 1A). Based on this information, rather than on Kabat definitions,it was decided which residues to take from the 4D5 framework and whichones to take from the MOC31 sequence. The resulting graft did thus notstrictly follow the CDR definition according to Kabat et al. (1979) orChotia (see Allazikani et al., 1997), but includes two residues (L64 andL66) which determine the conformation of the “outer loop” of V_(L)(residues L66-L71). The tip of this loop was shown to contact theantigen in some complexes, and an influence of this loop on theconformation of CDR L1 could not be excluded. Residue L66 usually is Glyin kappa light chains and assumes a positive φ angle. If this residue isreplaced by a non-Gly residue (Arg in 4D5), the outer loop assumes adifferent conformation, bending away from the domain. In V_(H), inaddition to CDR H1, residues H27 to H30 were included, while someresidues at the base of CDR2 were omitted (H62 and H63), despite beingpart of CDR H2 according to CDR defintions (Kabat et al. (1979);Allazikani et al., 1997), and several residues in the “outer loop” ofV_(H), sometimes referred to as CDR4, were included (residues H69, H71,H75-H77), resulting in the construct 4D5MOC-A (FIG. 1A).

Analysis of the conformations of V_(H) domain frameworks revealed thatthese can be classified according to their framework conformation into 4distinct subgroups. The conformational differences are most noticeablein framework 1 (FR1), particularly in positions H7–H10, althoughcorrelated sequence and conformational differences are found throughoutthe molecules, involving several core residues³¹. These conformationalchanges are probably caused by the different hydrogen bonding patternswhich the fully buried Glu H6 (as in 4D5) or Gln H6 (as in MOC31)establishes in the core of the domain, and are further influenced by thenature of residue H9 (Pro, Gly or other residues)³². Saul and Poljak(1993) reported correlated structural changes affecting residues H9,H18, H82, H67 and H63 which relay the effects of changes in FR1conformation across the domain core to the base of CDR2, thuspotentially enabling them to potentially affect antigen binding.

According to this classification, MOC31 belongs to a different subclassthan 4D5. Since we did not know to what extent these framework classesaffect the functionality of a loop graft, we decided to test this aspectexperimentally. While in construct 4D5MOC-A the V_(H) domain frameworkwas changed to the 4D5 subtype, 4D5MOC-B fully retains the MOC31 corepacking as well as the conformationally critical residues H6 and H9. Toachieve this, eight additional framework residues of the anti-EGP-2single-chain Fv fragment sequence (H6, H9, H18, H20, H38, H63, H82 andH109) had to retain the MOC31 sequence. All of these changes are locatedin the lower half of the scFv (FIG. 1), and with the exception of theGly to Pro substitution in position H9, are buried in the core of thedomain. They are therefore not expected to affect the immunogenicity ofthe construct.

For introduction of the AflII restriction site it was necessary tomodify the C-terminal sequence of the V_(L)-domain in all constructsfrom EIKRA to ELKRA, which should not affect the domain structure.

Expression and Purification of scFv Constructs

For scFv 4D5 usually 1–2 mg pure protein could be purified from oneliter of culture, while for scFv MOC31 the yield was much lower. Aftertwo steps of purification scFv MOC31 yielded only 200 μg at a purity ofabout 70%. Coexpression of skp³³ increased the yield to 600 μg, butthere was still a 20 kDa degradation product present. The graft variantscFv 4D5MOC-A could be purified to a yield of 400 μg and 4D5MOC-B to 1mg at a purity over 95%. Both single-chains Fv fragments could beconcentrated to 1 mg/ml and were analyzed on a reducing SDS-PAGE (FIG.2). Mass spectrometry of both molecules showed the expected molecularweight of 29,855 Da for scFv 4D5MOC-A and 29,897 Da for scFv 4D5MOC-B.

Binding Specificity

The transfer of the anti-EGP-2 binding specificity of scFv MOC31 ontothe framework of scFv 4D5 was shown to be succesful for both variants,4D5MOC-A and 4D5MOC-B, by binding competition of the radiolabelled graftvariants to EGP-2 overexpressing SW2 cells. Only the monoclonal antibodyMOC31 could inhibit binding of the graft variants, whereas an irrelevantcontrol antibody did not compete (FIG. 3A). No cross-reactivity of thegrafted molecules were seen when incubated on c-erbB2 or EGF-receptor(extracellular domain) (FIG. 3B).

Determination of K_(D)

High-affinity binding with long residual time on the specific targetantigen is regarded as one of the most important characteristics ofantibodies for tumor targeting. To ensure that binding affinity wasconserved in the grafting experiment dissociation constants of theradiolabelled single-chain Fv fragment were determined on cells in aradioimmunoactivity assay (RIA). The graft variants showed specific andsimilar binding behavior comparable to the parent anti-EGP-2single-chain Fv fragment in the nanomolar range (Table 1).

Binding kinetics of unlabelled scFv fragments to immobilized EGP-2 werealso analyzed by surface plasmon resonance (Table 1) in the BIAcoreinstrument (Pharmacia). To minimize rebinding effects which could leadto an underestimation of the off-rates, we used low density coating andhigh flow rate. ScFv MOC31 showed stable binding on its target with ahalf-life of about 38 min consistent with an independent determination(half-time of 33 min)³⁰. The k_(off) values of scFv 4D5MOC-A and4D5MOC-B were very similar to the parent scFv MOC31 (Table 1),indicating that the full transfer of the binding properties of scFvMOC31 on the 4D5-framework was successful.

Analytical Gel Filtration and Test of Thermal Aggregation

For many applications of scFvs it is crucial to concentrate thesemolecules and to incubate them at elevated temperatures. The biophysicalbehavior of these molecules is then often the threshold for theirapplicability in vivo. Therefore we tested the aggregation behavior ofthe scFvs at high concentrations and elevated temperatures. While 4D5,4D5MOC-A and 4D5MOC-B could be concentrated to 1 mg/ml byultrafiltration, the MOC31 scFv precipitated at concentrations above 400μg/ml. At this concentration, about 10% of the total protein eluted ashigh molecular weight aggregates on analytical gel filtration with theSmart gel filtration system (Pharmacia) on a Superdex 75 column. Almost90% of the protein eluted at a volume of 1.27 ml as expected for themonomeric species. However, already within 30 min at 37° C.,approximately 85% of the protein precipitated. The remaining 15% solubleprotein eluted as a monomeric species (data not shown).

The two grafted variants 4D5MOC-A and 4D5MOC-B eluted at a volume of1.20 ml, corresponding to a molecular weight of 30 kDa, indicating thatboth single-chain Fv fragments exist as monomers at a concentration of 1mg/ml. Although 4D5MOC-A precipitated more slowly than MOC31, overnightincubation in PBS at 37° C. for 20 hrs and subsequent gel filtrationshowed nearly no eluted protein (FIG. 4A). Incubated under the sameconditions, 4D5MOC-B still eluted as a symmetric peak at a volume of1.20 ml (FIG. 4B), indicating a large difference in intrinsic (thermal)stability of the two variants. Most importantly, 4D5MOC-B, was therebyshown to have the biophysical properties required for in vivoapplication.

His-tag Specific^(99m)Technetium-Labeling

The single-chain Fv fragments were labelled with ^(99m)Tc, using a newmethod in which ^(99m)Tc-tricarbonyl-trihydrate is stably bound to theC-terminal penta- or hexahistidine tag of recombinant proteins (Waibelet al, 1999). All scFv fragments, except the original scFv MOC31, couldbe labelled at 37° C. and at a protein concentration of 1 mg/ml,resulting in 30 –40% of the initial ^(99m)Tc incorporated, giving afinal specific activity of 300–400 mCi/ml. For the aggregation-pronescFv MOC31, the incubation temperature had to be lowered to 30° C. andthe maximal possible protein concentration was 400 μg/ml, resulting in adecreased incorporation yield (25% of total Tc, 250 mCi/ml).

Determination of Immunoactivity after Incubation in Serum at 37° C.

We determined the fraction of scFv molecules still active after ^(99m)Tclabeling (FIG. 4C)²⁹ and after incubation of the labelled fragments inhuman serum for 20 h at 37° C. (FIG. 4D). For scFv MOC31, we found67%±5.4 of the protein still active if the labeling reaction wasperformed at 30° C. The other fragments showed 47.25%±4.9 active forscFv 4D5MOC-A, 74.5%±8.3 for scFv 4D5MOC-B and 87.3%±6.4 for scFv 4D5,all labelled at 37° C. To test serum stability, the scFv fragments (17μg/ml) were incubated in human serum at 37° C. for 20 hours and theremaining immunoactivity determined. ScFv MOC31 was found to becompletely inactive after overnight incubation, therefore earlier timepoints were measured. Already after 1 h, the activity had dropped to6.32%±0.17 (9.4% of the initial immunoactivity). After 4 h, only1.95%±0.175 (2.9%) remained active. In contrast, the activity of4D5MOC-A dropped from 47.25%±4.9 to 8.1%±4.7 (17.1% of the initialvalue) over 20 h, that of scFv 4d5MOC-B from 74.5%±8.3 to 36%±1.6(48.3%) and that of scFv 4D5 from 87.3%±6.4 to 40.45%±8.75 (46.3% ),confirming the different thermal stabilities found in the gel filtrationassay.

In vivo Characterization—Clearance and Biodistribution

Biodistribution studies were then performed for scFv ^(99m)Tc-labelledscFvs MOC31, 4D5, 4D5MOC-A and 4D5MOC-B. For scFv MOC31 we were unableto get a tumor-to-blood ratio higher than 0.92 after 1 h, 4 h and 24 h(n=3, each time point). After 24 h the total dose at the tumor was 1.24%ID/g tissue, but also 1.34% ID/g in the blood, which was very high incomparison to the 3–5 fold lower values usually found in the blood after24 h with that labeling method (Table 2). In contrast, thebiodistribution of ^(99m)Tc labelled scFv 4D5 gave a tumor-to-bloodratio of 8.3 after 24 h with a total dose of 1.5% ID/g on SK-OV-3 cells(Waibel et al., 1999), similar results were reported for theanti-c-erbB2 scFv C6.5³⁴. For 4D5MOC-A we found after 24 h only a weakenrichment with a total dose of 0.84% ID/g and a tumor-to-blood ratio of1.95 (Table 3), while the in vivo application of scFv 4D5MOC-B inSW2-tumor-bearing mice resulted in a tumor-to-blood ratio of 5.25 after24 h with a total dose of 1.47% ID/g at the tumor. The maximal dose atthe tumor was measured after 4 h with 1.82% ID/g, which then decreasedvery slowly, reflecting fast and stable binding of scFv 4D5MOC-B to theantigen (Table 3). For the nonspecific anti-fluorescein control scFvFITC-E2¹⁵ no enrichment at the tumor site was found (Table 4).

Clearance studies revealed scFv 4D5MOC-B as a very rapid clearingmolecule with a t_(1/2)α=6 min and t_(1/2)β=228 min. The comparison withscFv 4D5 with a measured t_(1/2)(α)=7.5 min shows that the excellentclearing behavior, which is a prerequisite for the achievement of hightumor-to-blood-ratios is not lost by the loop grafting.

Discussion

It has been reported that indium-DTPA-labelled mAb MOC31 localized toprimary tumor and metastases in a clinical trial with small cell lungcancer patients, but it was not superior to other diagnostic methodse.g. computer-tomography scan⁴. A chemical fusion of mAb MOC31 with theexotoxin-A (ETA) led to growth delay for large tumors (120 mm³) in nudemice, and it was proposed that the reduction of the targeting antibodyin size would increase the efficiency⁸. It remains to be tested whetherthe improved tissue penetration and faster clearance rate of the muchsmaller anti-EGP2 scFv fragment will yield better results or whether itsincreased ability to access to normal EGP-2-expressing epithelialtissues, not accessible to mAbs due to their molecular weight of 150kDa¹, will limit the resolving power of the method. The improved scFvcan serve as a building block for other recombinant molecule formatssuch as dimerized and multimerized scFv, Fab or (Fab)² ³ to optimizesize- and avidity effects. They can also be fused to other effectordomains in the construction of antibody fragment based therapeutics. AscFv MOC31-ETA fusion was in vitro on SW2 cells ten thousand times moretoxic than the mab MOC31-fusion with ETA (Zimmermann, unpublishedresults). The original unmodified scFv MOC31 was also used for theconstruction of a diabody with CD3 specificity for T-cell retargeting.In this format scFv MOC31 appeared to be somehow stabilized and a halflife of 12 h has been reported, but the yield was as poor as for scFvMOC31 alone³⁷ and an in vivo application was not reported so far.

During the modelling we noticed that the V_(H) domain of the frameworktemplate 4D5 belongs to a different structural subclass than the loopdonor MOC31. Since there are several examples in the literature in whicha simple loop graft failed and the chimeras had to be rescued bymultiple additional back-mutations³⁶, we directly designed a secondchimera in which the structural subclass and core packing of MOC31 wasretained. This involved changing of eight additional residues, mostly inthe core, to the murine sequence, than essentially corresponding to aresurfacing of the MOC31 V_(H) domain. These additional mutations had noeffect on antigen affinity, but they had a beneficial effect on thestability of the chimera. The additional mutations in 4D5MOC-B yielded amolecule which behaved very similar to the very well behaved 4D5 scFv.This is remarkably, as 4D5MOC-B is further removed in sequence from 4D5than 4D5MOC-A and suggests that it may be critical to maintain aframework class, as defined by residues H6, H7 and H9 throughout and notmix the framework as these residues are interrelated. Furthermore, while4D5MOC-B is closer in sequence to MOC31, the latter molecule is theleast stable of all.

It has recently been shown that the V_(L) domain of 4D5 is exceptionallystable and the thermodynamic stability of the 4D5 scFv is limited byintrinsic stability of its V_(H) domain²⁰. Grafting of the MOC31 antigeninteraction surface onto this fragment resulted in a chimera ofintermediate stability. This could be due to unfavorable interactionswithin the grafted loops or between grafted core residues andincompatible framework core residues. However, there are few contactsbetween those framework residues in the lower core which differ between4D5 and MOC31 and core residues from the grafted loops, the two beingseparated by a layer of conserved residues (FIG. 1). The main directcontact between the residues changed in the loop graft and the group ofresidues additionally changed in 4D5MOC-B is between Met H48 (Val in4D5) and Phe H63 (Val in 4D5 and in 4D5MOC-A). If there had been asteric clash in the original graft, we would have expected the situationto be aggravated by the substitution of the contact residue with alarger residue. It is therefore more likely that the destabilizinginfluence of the loops has been compensated by a general stabilizationof the domain core.

The additional stabilization achieved by the core mutations in 4D5MOC-Bwas of crucial importance for effective enrichment at the tumor site.The most stable construct, 4D5MOC-B, enriched to 1.47% ID/g tissue witha tumor-to-blood ratio of 5.25. The aggregation-prone MOC31 was clearedfrom the circulation much more slowly than the more stable controlantibodies and chimeras. It remains to be seen to what extent a furtherincrease in stability can further improve the total dose enrichment andtumor-to-blood ratios.

We demonstrate in this study that the strategy of engineering forfolding and stability is general tool for the improvement of interestingantibody-fragments. We used as an example the conversion of an unstableand poorly expressing murine anti-EGP-2 scFv, which failed in vivo, to awell expressing and very stable humanized antibody fragment of the samespecificity. E also report in vivo targeting of EGP-2 presentingxenografts in CD1 nude mice for the first time. The engineered scFv4D5MOC-B overcomes the limitations of scFv MOC31 and will be animportant a building block for the development of new imaging andtherapeutic antibody fragment-based reagents, directed to EGP-2expressing carcinomas. We believe that in addition to the use of largelibrary repertoires from which new antibody fragments with outstandingproperties can be selected, the engineering for folding and stability ofrecombinant molecules is of extraordinary importance for theirwidespread future use in all applications, and especially those in tumormedicine. It must be emphasized again that biophysical propertiesstrongly influence the ability of a scFv fragment to target to a tumorsite, even when the complementary determining regions and the bindingconstants are identical. This indicates that the biophysical propertiesof an antibody fragment have a far greater importance for the biologicalperformance than has been generally appreciated up to now.

TABLE 1 Comparison of affinities and kinetic rate constants asdetermined by radioimmunoassay (RIA) on SW2 cells and surface plasmonresonance (BIAcore) RIA Surface plasmon resonance (SPR) on SW2 cellsk_(on) k_(off) Antibody Kd* (nM) Kd^(Δ)(nM) (10⁵M⁻¹s⁻¹) (10⁻³s⁻¹)scFvMOC31 10.8 ± 2.6  3.0 0.99 ± 0.01  0.3 ± 0.01 4D5MOC-A 3.6 ± 0.5 3.5 1.29 ± 0.001  0.45 ± 0.001 4D5MOC-B 3.7 ± 0.5 3.9 1.84 ± 0.02 0.717 ±0.001 Measurements were performed at 4° C. (*) and 20° C. (Δ)

TABLE 2 Biodistribution of 99mTc-labeled scFvs: in Balb/C-nude micexenografted with SW2-tumors ScFv MOC31 FITC-E2 1 h (n=3) 4 h (n=3) 24 h(n=3) 24 h (n=3) Organs % ID/g % ID/g % ID/g % ID/g Blood 8.46 ± 0.875.55 ± 1.98 1.34 ± 0.15  0.5 ± 0.13 Heart  5.1 ± 0.34 5.32 ± 1.36 1.39 ±0.25 0.47 ± 0.23 Lung 7.37 ± 0.95 7.14 ± 1.61 2.09 ± 0.5  0.58 ± 0.16Spleen 14.86 ± 1.91  17.84 ± 2.77   7.6 ± 0.56  1.2 ± 0.09 Kidney 253.69± 10.64  263.28 ± 43.1  117.4 ± 12.2  224.09 ± 40.4  Stomach 4.71 ± 1.044.08 ± 1.04 1.28 ± 0.21 0.25 ± 0.14 Intestine 5.85 ± 0.34  5.4 ± 1.971.56 ± 0.07 0.34 ± 0.04 Liver 35.03 ± 0.86  44.8 ± 9.54 20.38 ± 3.44 4.44 ± 0.65 Muscle 1.64 ± 0.23 1.57 ± 0.36 0.75 ± 0.82 0.31 ± 0.15 Bone5.97 ± 0.41 6.01 ± 1.71 2.04 ± 0.68 0.57 ± 0.3  Tumor 2.46 ± 0.88 3.97 ±1.07 1.24 ± 0.74 0.4 ± 0.2 Tumor-to- 0.29 0.71 0.92 1.35 blood ratioBiodistribution of 99mTc-labeled scFv MOC31 and FITC-E2 was studied ineight-week-old female Balb/C nude mice which bear 17 days old SW2-tumorsafter injection of the radiolabeled antibodies into the animals. Thenumbers represent % injected dose per gramm tissue (% ID/g). The resultsare expressed as the mean.

TABLE 3 Biodistribution of ^(99m)technetium-labeled scFvs: in CD1-nudemice xenografted with SW2-tumors 4D5MOC-B 4D5MOC-A FITC-E2 1 h (n=3) 4 h(n=3) 24 h (n=3) 24 h (n=3) 24 h (n=3) Organs % ID/g % ID/g % ID/g %ID/g % ID/g Blood 2.92 ± 0.47 1.31 ± 0.23 0.28 ± 0.06 0.43 ± 0.20 0.17 ±0.02 Heart 0.97 ± 0.21 0.57 ± 0.11 0.28 ± 0.09 0.63 ± 0.18 0.16 ± 0.04Lung  3.2 ± 1.29  1.2 ± 0.08 1.14 ± 0.60 1.77 ± 0.95 0.24 ± 0.05 Spleen0.61 ± 0.06 0.67 ± 0.19  0.7 ± 0.13 1.57 ± 0.44 0.22 ± 0.04 Kidney120.79 ± 7.19  140.56 ± 3.94  300.17 ± 85.2  90.53 ± 52.4  383.91 ±57.3  Stomach 0.48 ± 0.09 0.49 ± 0.1  0.24 ± 0.21 0.26 ± 0.07 0.26 ±0.13 Intestine 1.33 ± 0.64 0.71 ± 0.06 0.30 ± 0.07 0.48 ± 0.15 0.21 ±0.07 Liver 6.49 ± 1.53 6.86 ± 0.32 2.38 ± 0.52 4.37 ± 1.87 1.33 ± 0.33Muscle 0.27 ± 0.01 0.17 ± 0.03  0.1 ± 0.02 0.21 ± 0.09 0.07 ± 0.01 Bone0.29 ± 0.21 0.21 ± 0.16 0.06 ± 0.05 0.25 ± 0.31 0.06 ± 0.05 Tumor 1.74 ±0.51 1.82 ± 0.22 1.47 ± 0.32 0.84 ± 0.38 0.23 ± 0.04 Tumor-to- 0.59 1.385.25 1.95 1.35 blood ratio Biodistribution of ^(99m)Tc-labeled scFv4D5MOC-A, 4D5MOC-B and FITC-E2 was studied in eight weeks old female CD1nude mice which bear 13 days old SW2-tumors after injection of theradiolabeled antibodies into the animals. The numbers represent %injected dose per gramm tissue (% ID/g). The results are expressed asthe mean.

LITERATURE

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1. A method for stabilizing a chimeric immunoglobulin or immunoglobulinfragment (chimera) that comprises i) VH domain antigen-binding loopsfrom a donor immunoglobulin or immunoglobulin fragment (donor) which isable to bind to an antigen, and ii) VH domain framework regions from anacceptor immunoglobulin or immunoglobulin fragment (acceptor), whereinthe VH domains of said donor and of said acceptor belong to differentframework structure subgroups, and wherein said method comprises thestep of replacing in the chimera one or more of the residues present atidentified framework positions in the acceptor by those present atcorresponding positions in the donor, wherein different amino acidresidues present in said acceptor and said donor at one or more of saididentified framework positions lead to the formation of differentframework structure subgroups, wherein said identified frameworkpositions are selected from the group consisting of H6, H7, H8, H9, H10,H18, H20, H38, H63, H67, H82 and H109 in said VH domains, wherein astabilized antigen-binding immunoglobulin or immunoglobulin fragment isestablished, and wherein said chimera is able to bind to said antigen.2. A method for stabilizing a chimeric immunoglobulin or immunoglobulinfragment (chimera) that comprises i) VH domain antigen-binding loopsfrom a donor immunoglobulin or immunoglobulin fragment (donor) which isable to bind to an antigen, and ii) VH domain framework regions from anacceptor immunoglobulin or immunoglobulin fragment (acceptor), whereinthe VH domains of said donor and of said acceptor belong to differentframework structure subgroups, and wherein said method comprises thesteps of a) identifying one or more framework positions in the aminoacid positions selected from the group consisting of H6, H7, H8, H9,H10, H18, H20, H38, H63, H67, H82 and H109 in said VH domains wheredifferent amino acid residues present in said acceptor and said donorlead to the formation of said different framework structure subgroups;and b) replacing in the chimera one or more of the residues present atsaid identified framework positions in the acceptor by those present atcorresponding positions in the donor, wherein a stabilizedantigen-binding immunoglobulin or immunoglobulin fragment isestablished, and wherein said chimera is able to bind to said antigen.3. The method of claim 1 or claim 2 wherein said different amino acidresidues present in said acceptor and said donor at one or more of saididentified framework positions that lead to the formation of differentframework structure subgroups are identified by analyzing VH domainstructures and/or structure models.
 4. The method of claim 1 or claim 2wherein one of said identified framework positions is H6.
 5. The methodof claim 1 or claim 2 wherein one of said identified framework positionsis H9.
 6. The method of claim 1 or claim 2 wherein said acceptor is thehuman anti-c-ErbB2 scFv fragment 4D5 (SEQ-ID No. 1).
 7. The method ofclaim 6 wherein said donor is the anti-EGP-2 scFv fragment obtained fromthe murine hybridoma MOC31 (SEQ-ID No. 2).
 8. The method of claim 7wherein said identified framework positions comprise H6, H9, H18, H20,H38, H63, H82 and H109.
 9. The method of claim 8 wherein said stabilizedantigen-binding immunoglobulin or immunoglobulin fragment is theanti-EGP-2 scFv fragment 4D5MOC-B (SEQ-ID No. 3).
 10. The method ofclaim 1 or claim 2, wherein said chimera additionally comprises furtherresidues from said donor for improving antigen-binding.